The availableness of a large number of biological reagents, such as hundreds of thousands of deoxyribonucleic acids (DNA) clones, numerous antibodies and recombinant proteins, millions of compounds obtained through combinatory chemical synthesis, has promoted the development of technologies for high throughput studies of these molecules. Special arrays of biological reagents have been designed, in which each of the reagents is placed at a pre-defined position and can be identified later by the position. These arrays of biological reagents have found a wide variety of applications. Protein arrays have been applied in studying protein expression patterns, protein posttranslational modifications such as phosphorylation, glycosylation, lipidation and ubiquitination. Protein arrays are also used in screening protein-protein interactions (Wang et. al., Mol. Cell Biol. 20, 4505-12). Arrays of nucleic acids are used for large scale hybridization assays, including monitoring of gene expression (Schena et al., 1995, Science 270:467-470; DeRisi et al., 1996, Nature Genetics 14:457-460), genetic and physical mapping of genomes, genetic diagnosis, genotyping of organisms, detection of DNA-protein interactions (Bulyk et al. Nature Biotechnology, 17:573-577, 1999), and distribution of biological reagents to researchers (see U.S. Pat. No. 5,807,522). DNA arrays are also used to obtain nucleotide sequence information, including mutation detection, polymorphism detection and DNA sequencing (Hacia, Nature Genetics Volume 21, supplement, p42-47, 1999). In addition, arrays of cells, tissues, lipids, polymers, drugs or chemical substances can be fabricated for large scale screening assays in medical diagnostics, drug discovery, molecular biology, immunology and toxicology (see Kononen J, et al., Nature Medicine, 4:844-7, 1998).
A variety of methods are currently available for making arrays of biological reagents, such as arrays of nucleic acids and proteins. One method for making an ordered array of DNA on a porous membrane is a “dot blot” approach, in which a plurality of DNA in solutions are transferred by vacuum to a porous membrane. A common variant of this procedure is a “slot blot” method in which the wells have highly elongated oval shapes. A more efficient way for making ordered arrays of molecules uses an array of pins dipped into the wells, e.g., the 96 wells of a microtitre plate, for transferring an array of samples to a substrate, such as a porous membrane. The pins can be designed to spot a membrane in a staggered fashion, for creating an array of thousands of spots in a small area (see Lehrach, et al., Hybridization fingerprinting in genome mapping and sequencing, genome analysis, Vol 1, Davies and Tilgham, Eds, Cold Spring Harbor Press, pp. 39-81, 1990). Recently Brown et al. (U.S. Pat. No. 5,807,522) described a more elaborated method to make arrays. The method involves dispensing a known volume of a reagent at each selected array position, by tapping a capillary dispenser on the support under conditions effective to draw a defined volume of liquid onto the support. An alternate method of creating ordered arrays of nucleic acid sequences was described by Fodor, et al. (Science, 251: 767-773, 1991). The method involves synthesizing different nucleic acid sequences at different discrete regions of a support, usually made of glass. A related method was described by Matson, et al. (U.S. Pat. No. 5,429,807, 1995). A method of making arrays of polypeptides by photolithographic solid phase synthesis was described by Pirrung, et al. (U.S. Pat. No. 5,143,854, 1992).
Since in prior arts, arrays of biological reagents are mainly used in binding assays, such as DNA-DNA hybridization, DNA-RNA hybridization, DNA-protein binding, RNA-protein binding and protein-protein binding, DNA and protein arrays are accordingly fabricated for the purpose of performing these assays. For example, in dot blot or slot blot method, DNA are usually immobilized by baking or by exposing to UV radiation; and in DNA Chip manufactured by Affymatrix, oligo nucleotides are synthesized on glass supports through covalent bonds. Strong immobilization through covalent or multi-valent non-covalent bonds is necessary for binding or hybridization assays, which require extended incubations and multiple washes. However, covalent or very strong non-covalent immobilization used in making DNA or protein arrays by previous methods is not suitable for some other potential applications. For example, it is difficult to introduce DNA covalently bond to a support into cells. Therefore, the support materials and immobilization methods in prior arts are not suitable for introducing a large number of DNA or proteins into cells. The applications of DNA and protein arrays are thus severely limited. New techniques are needed to make arrays that can be used not only for binding assays but also for other applications, such as transfecting cells with arrays of DNA or proteins; and staining cells with arrays of antibodies.
Transfection is in general term the method to introduce biological reagents into target cells. The biological reagents, such as proteins, DNA and ribonucleic acids (RNA), are normally unable to cross cell membranes and enter cells. Transfection usually includes the steps of contacting the target cells with the reagents to be transfected, applying a condition such as an electric field to make cells uptake the reagents. There are many methods for transfection and they are referred by different names in prior arts. Transformation sometimes refers to the process of introducing a piece of DNA, usually in a vector, into bacteria. Infection is the process to deliver nucleic acids into cells by viruses. Numerous cell types have been transfected, which include bacteria, yeast, plant cells, insect cells, mammalian cells and human cells. Cells from a given source, e.g., a tissue, or an organ, or cells in a given state of differentiation, or cells associated with a given pathology or genetic makeup can be transfected.
Transfection of biological reagents into cells has a variety of applications. One of them is to study the functions of DNA and proteins. For examples, if introduction of an antibody against a protein into cells causes the cells to behave abnormally, then the function of the protein can be inferred from this abnormal phenotype. Likewise, after introducing an exogenous gene into a cell line, one can study the effects of the gene on cell growth, cell death and other cell behaviors. The regulation of the transfected gene, either its expression or activity, can also be studied.
Another application of transfection is to isolate genes of interest. A standard protocol for this application involves transfecting cells with a pool of DNA; selecting the transfected cells with a desired phenotype and recovering the DNA from the cells. Such techniques include but are not limited to expression cloning, complementary DNA (cDNA) libraries screening, expression library screening (see Sambrook et al., Molecular Cloning, a laboratory manual, Cold Spring Harbor Press, 1989) and yeast two-hybrid screening (see U.S. Pat. No. 5,283,173). Many genes encoding ion channels, membrane receptors and signaling proteins have been isolated using these techniques.
Transfection is also a key step in producing large quantities of nucleic acids and proteins. For example, transformation has long been used to propagate and amplify DNA in bacterial host. By introducing a gene into bacteria, large quantities of the protein encoded by the gene can also be produced. The proteins thus obtained are valuable for both research and therapeutic applications.
By stably expressing an exogenous gene into cells, one can change the properties and functions of the cells. These cells may then be used for therapeutic applications. For example, somatic cells removed from a patient with a defective gene can be transfected with a correct version of the same gene. Replacement of the transfected cells back into the patient may improve the patient's condition. This approach has been particularly successful in introducing genes into lymphocytes. Examples of transfection for gene therapy and some other applications can be found in publications by Bordignon et al. and Dick et al. (Science 270:470-475, 1995 and Blood 78:624-634, 1991 respectively) which are hereby incorporated by reference.
Biological reagents may be introduced into prokaryotic cells and some eukaryotic cells with varying degrees of ease. For example, heat shock method is routinely used to transfect DNA into bacteria and yeast. However, it is more difficult to introduce DNA into eukaryotic cells, such as human cells. Some sophisticated methods have been designed for this purpose and many improvements are used to increase transfection efficiency.
One way to introduce biological reagents into cells is by direct microinjection. Although it is difficult to introduce reagents into a large number of cells by this method, microinjection is useful for delivering reagents into some special cells, such as oocytes, skeletal muscles and neurons, which may be resistant to other transfection methods. Microinjection is also valuable when the number of target cells available is limited.
Biological reagents can also be introduced into cells by particle bombardment. In this method, microscopic particles, coated with the reagents to be transfected, are accelerated by a shock wave in a gaseous medium so that the particles are able to penetrate cells and deliver the reagents thereto. The shockwave may be produced by a variety of means including high-voltage electrical discharge (see McCabe et al., Bio/Technology 6, 923, 1992; U.S. Pat. No. 5,149,655) or helium pressure discharge (see Williams et al., Proc Natl Acad Sci USA 88, 2726, 1991).
DNA uptake by cells can be enhanced by facilitators such as calcium phosphate and diethylaminoethyl (DEAE)-dextran. Treatment with either of these chemicals is thought to produce an environment that promotes the attachment of DNA (presumably in complex with either calcium phosphate or DEAE-dextran) to the cell surface and subsequent endocytosis. DEAE-dextran is especially useful for transient transfection (Gonzalez A. L., et al. Trends Genet. 11:216-7, 1995). The original protocol for calcium phosphate transfection was described by Graham and van der Eb (Virology, 52: 456-467, 1973). This method was modified by Wigler et el. (Proc. Natl. Acad. Sci., 76: 1373-1376, 1979) and by Chen and Okayama (Mol. Cell. Biol., 7: 2745-2752, 1987).
Artificial membrane vesicles (liposomes) are useful delivery vehicles in vitro and in vivo. Most of these procedures involve encapsulation of DNA or other molecules with liposomes, followed by fusion of the liposomes with the cell membranes (Hofland, H. E .J., et al., Proc. Natl. Acad. Sci. USA 93: 7305-7309, 1996; Gao, X., and Huang, L., Biochem. 35:1027-1036, 1996; Liu, Y., et al., J. Biol. Chem. 270: 24864-24870, 1995). DNA are usually complexes with cationic substances, which may include cationic lipids, cationic polyamino acids (e.g., poly-L-lysine and polyomithine), cationic amphiphiles and polyethyleneimine. Examples of using cationic lipids for transfection are found in U.S. Pat. Nos. 5,616,745 and 5,851,818, which are hereby incorporated by reference.
Application of one or several short and sufficiently strong electric pulses to a suspension or monolayer of target cells may break down some parts of the cell membranes to form minute pores. Surrounding molecules can then diffuse or are driven into the target cells during the time when the cell membranes remain permeable to these molecules. This process is called electroporation. Methods of using electroporation to transfect cells can be found in the publication by Shigekawa and Dower (BioTechniques, Vol. 6: 742-751, 1988) and U.S. Pat. Nos. 4,910,140 and 4,750,100; which are hereby incorporated by reference. Electroporation is used to transfect both cells in suspension and cells adhering on a solid support. The method of electroporating cells adhering on a solid support can be found in Yang, et al., Nucleic Acid Research, Vol. 23, p2803-2810, 1995; and in Firth et al., BioTechniques 23:644-646, 1997.
Viruses derived from different sources are used for introducing genes into target cells. For example, bacterial phages have long been used in making DNA libraries in bacteria. Several widely used viral vectors for gene transfer into mammalian cells are derived from retroviruses (Miller, A. D., 1990, Human Gene Ther. 1:5-14) and adenovirus. Adenovirus vectors have been utilized for gene therapy and for gene expression in highly differentiated cells such as neuronal cells. Viral particles can be deposited on a solid support to increase the contacts between the particles and target cells, and thus the infection efficiency (see U.S. Pat. No. 5,811,274.).
In conventional methods, transfection is usually performed to deliver a homogenous biological reagent into one type of homogenous cells. Even if transfection is performed to introduce more than one reagent into cells, after transfection, the cells that contain a specific reagent are not known without further identification. One such example is the preparation of cDNA, genomic or expression libraries in bacteria. Screening is required to identify the cells expressing a specific DNA or protein. Library screening is feasible for identifying one or few cell groups, each of which expresses a reagent of interest. But when the effects of many transfected reagents on the cells are to be studied, the conventional methods are inadequate. A method is therefore needed to transfect multiple reagents into cells in such a manner that the cells containing each of the transfected reagents can be quickly and easily identified and examined.
Cell staining is a versatile technique widely used in research and diagnostics to demonstrate the presence of specific antigenic determinants on cells or tissues and to quantify the numbers of cells bearing particular determinants in a heterogeneous population (See Harlow and Lane, Antibodies, a laboratory manual, Cold Spring Harbor Press, 1988). The first step in a standard staining protocol is to attach cells to be stained to a solid support. Adherent cells may be grown on microscope slides, coverslips, or other optically suitable materials. Suspension cells can be handled in a suspension or centrifuged onto a solid support and bound to the support using chemical linkers. The second step is to fix and permeabilize the cells to expose the antigen. The cell preparations are then incubated with antibodies and washed to remove unbound antibodies.
Recently developed tissue arrays allow the staining of many different cells with one or few (usually no more than two) different antibodies (Kononen J, et al., Nature Medicine, 4:844-7, 1998). For some other applications, such as to screen proteins having a particular subcellular localization, cells must be stained with antibodies against a large number of different proteins. Most of the current methods only allow cell staining with less than a few antibodies at a time. Therefore a new method of staining cells is needed for such purposes.